Jak-STAT signaling in liver disease and repair : a way of living

J AK -STAT SIGNALING IN LIVER DISEASE AND REPAIR

a Way of living

Inauguraldissertation zur

Erlangung der Würde eines Doktors der Philosophie vorgelegt der

Philosophisch-Naturwissenschaftlichen Fakultät der Universität Basel

von

Simone Tjitske Dorothea Stutvoet

aus Apeldoorn, die Niederlande

Basel, 2005

Genehmigt von der Philosophisch-Naturwissenschaftlichen Fakultät auf Antrag von

Prof. Dr. phil. H.P.Hauri Prof. Dr. med. M.H.Heim Prof. Dr. med. U.A. Meyer Basel, den 28. September 2004

Prof. Dr. M. Tanner

Dekan der Philosophisch-

Naturwissenschaftlichen Fakultät

Such knowledge is a wonder greater than my powers;

it is so high that I may not come near it.

Ps.139:6

.

Contents

Abbreviations 7

Abstract of this thesis 9

Chapter 1 General Introduction 11

1.1 Jak-STAT signaling 12

1.1.1 Ligands and their receptors 12

1.1.2 Janus kinases (Jaks) 14

1.1.3 STATs: structure and functional domain 14 1.2 Mechanisms and regulation of Jak-STAT signaling 17

1.2.1 IFN signaling 17

1.2.2 IL-6 signaling 20

1.2.3 Nucleocytoplasmic transport 21 1.2.4 STAT DNA binding and transcriptional

activation 22

1.2.5 Negative regulation 23

1.2.6 Specificity and diversity in signaling 24 1.3 Biological functions of STAT proteins 25 1.3.1 Impact of STATs in disease 28

1.4 Hepatitis C virus (HCV) 29

1.4.1 Genomic organization of HCV and functions

of viral proteins 29

1.4.2 HCV polyprotein processing and replication 31 1.4.3 Models to study HCV escape of immune

response 32

1.5 Liver cell regeneration 33

1.5.1 Liver 33

1.5.2 Regulation of liver regeneration 33

1.6 Aim of this thesis 35

Chapter 2 Expression of Hepatitis C virus proteins inhibits

Interferon-alpha signaling in the liver of transgenic mice 37

Chapter 3 Expression of Hepatitis C virus structural proteins inhibits Interferon-alpha induced signaling through

the Jak-STAT pathway 55

Chapter 4 A fusionprotein between wild type murine STAT3 and the modified ligand-binding domain of the estrogen receptor can

mimic activated STAT3 in the presence of 4-hydroxytamoxifen 73

Chapter 5 Generation of in vivo conditional gain-of-function models to

study the role of STAT3 and STAT5a signaling in liver 93

Chapter 6 Outlook on the use of the conditional albmSTAT3wt-ER and

albmSTAT5aR618K-rER gain-of-function mice models 113

References 118

Acknowledgments 134

Curriculum vitÆ 138

7 Abbreviations

4-HT 4-hydroxytamoxifen

aa amino acids

AFP alpha-fetoprotein Alb albumin promoter ALT alanine aminotransferase BAC bacterial artificial

chromosome

Cdc cell-division-cycle (gene) Cdk cyclin-dependent kinase CTL cytotoxic T lymphocytes DMEM Dulbecco’s minimum essential

medium

EDTA ethylene-diamine-tetraacetic

acid

EGF epidermal growth factor EGTA ethylene glycol tetraacetic acid eIF2α eukaryotic translation

initiation factor-2 alpha EMSA electrophoretic mobility shift assay

ER estrogen receptor

Frt FLPe recombinase target G6PC glucose 6-phosphatase GAS gamma interferon activated

sequence

GH growth hormone

gp-130 glycoprotein 130

GTP guanosine triphosphate HCC hepatocellular carcinoma HCV hepatitis C virus

HGF hepatocyte growth factor

His histidine

HR homologous region

ICE interleukin-1β converting enzyme

IFN interferon

IFNAR interferon alpha receptor IFNGR interferon gamma receptor

Ig immunoglobulin

IH immunohistochemistry

IL-6 interleukin-6

IL-6R interleukin-6 receptor IP immunoprecipitation IRES internal ribosomal entry site IRF interferon regulatory factor ISG interferon-stimulated gene ISGF3 interferon-stimulated gene

factor 3

ISRE interferon-stimulated

response element

Jak Janus kinase

KO knock-out

LBD ligand binding domain

LCMV lymphocytic choriomeningitis virus

LIF leukemia inhibitory factor LPS lipopolysaccharide

MAPK mitogen-activated protein kinase MEF mouse embryonic fibroblasts MHC major histocompatibility complex Na3VO4 sodium orthovanadate

NFκB nuclear factor κB NP-40 nonidet P40

NS non-structural

NTR non-translated region

OAS 2’-5’-oligoadenylate synthetase ORF open reading frame

PAC P1-derived artificial chromosome PBS phosphate-buffered saline PCK-1 phosphoenolpyruvate

carboxykinase-1

PCR polymerase chain reaction PFGE pulsed-field gel electrophoresis Pfu plaque-forming units PH partial hepatectomy

PIAS protein inhibitor of activated STAT PKR double-stranded RNA-activated

protein kinase

PMSF phenylmethylsulfonyl fluoride PP2Ac protein phosphatase 2A catalytic

subunit

PRMT1 protein arginine methyl- transferase PTK protein tyrosine kinase PTP protein tyrosine phosphatase rER rat estrogen receptor

RT room temperature

RT-PCR reverse-transcriptase PCR SAP serum amyloid P

SDS-PAGE sodium dodecyl sulfate

polyacrylamide-gel electrophoresis

Ser serine

SH2 src-homology 2

SHP1 SH2-containing tyrosine

phosphatase 1

SIF serum inducible factor

SOCS suppressor of cytokine signaling STAT signal transducer and activator of

transcription

SUMO small ubiquitin-related modifier TAD transcriptional activation domain TC-PTP T-cell protein tyrosine

phosphatase TGF transforming growth factor TNF tumor necrosis factor

Tyr tyrosine

VEGF vascular endothelial growth factor VSV vesicular stomatitis virus

9 Abstract of this thesis

Signaling through the Jak-STAT pathway is initiated when an extracellular signaling protein binds to its corresponding transmembrane receptor. This leads to activation of the Jaks. The Jaks mediate phosphorylation at the specific receptor tyrosine residues, which then serve as docking sites for the STATs. Once recruited to the receptors, the STATs become phosphorylated by the Jaks. Activated STATs will dimerize, translocate to the nucleus and target gene promoters. STAT proteins display a wide range of functions in many biological aspects. Consequently they are involved in or mediating pathological processes.

This thesis describes and discusses our research, performed to understand in more detail the role of Jak-STAT signaling in liver pathophysiology.

Hepatitis C virus (HCV) is a major cause of chronic liver disease. It is believed that HCV proteins interfere with interferon alpha induced Jak-STAT signaling in order to escape the interferons induced antiviral state. We therefore analyzed interferon alpha induced Jak-STAT signaling in the presence of HCV viral proteins in in vivo and in vitro models to gain more insight in this interference, as well as to identify the viral protein(s) responsible for this interference. In chapter 2 we show inhibition of Jak- STAT signaling in transgenic mice that express HCV proteins in their liver cells. The inhibition occurred in the nucleus and blocked binding of STATs to the promoters of interferon stimulated genes. This inhibition of interferon induced signaling resulted in an enhanced susceptibility of the HCV transgenic mice to LCMV infection and the development of severe hepatitis. The results described in chapter 3, show that the combined HCV structural proteins and the core protein alone partly inhibit interferon alpha induced Jak-STAT signaling, using a panel of tetracycline-regulated cell lines inducibly expressing individual HCV proteins or in different combinations. In cells expressing HCV nonstructural proteins, interferon alpha induced Jak-STAT signaling was not impaired.

The last chapters of this thesis describe the generation of conditional active gain- of-function models to study STAT signaling independent of natural ligands. Fusion proteins were constructed between STAT1, STAT3 or STAT5a and the modified ligand binding domain of the estrogen receptor (ER) and subsequently expressed in mouse embryonic fibroblasts. In addition several mutants of the fusion proteins were generated. The fusion protein between wild-type STAT3 and the ER (mSTAT3wt-ER) was shown to bind DNA and mimic STAT3 signaling upon activation by 4- hydroxytamoxifen (synthetic steroid ligand) only (chapter 4). mSTAT3wt-ER and mSTAT5aR618K-rER (SH-2 domain mutant) fusion protein constructs were used to generate liver-specific conditional active gain-of-function mouse models.

Chapter 5 describes the multistep cloning approach to prepare the constructs for injection and the analysis of the transgenic mice designated albSTAT3wt-ER and albSTAT5R618K-rER. The transgenic mice were designed to express the fusion proteins under control of the albumin promoter allowing liver-specific expression.

Transgenic founders have been identified, as well as a transgenic F1 offspring. The transgenic mice appeared to be healthy and show a normal phenotype. Further characterization has to be completed. In chapter 6 possibilities for future use of the in chapter 5 described transgenic mice are discussed. The mice could be used to study STAT3 and STAT5a signaling in processes as liver cell regeneration, cellular transformation, bacterial infection, gluconeogenesis and cell survival.

Chapter 1 General Introduction

12 1.1 Jak-STAT signaling

The Janus kinase (Jak)-signal transducer and activator of transcription (STAT) pathway is an intracellular signal transduction pathway that transmits information received from extracellular signaling proteins (cytokines, growth factors and hormones) through transmembrane receptors to target gene promoters in the nucleus ¹. Binding of the ligand to its corresponding receptor leads to conformational changes in the cytoplasmic part of the receptor, allowing homo- or heterodimerization of the receptor subunits and initiating signaling through tyrosine phosphorylation of the receptor associated members of the Jak family of protein tyrosine kinases. The activated Jaks mediate phosphorylation at the specific receptor tyrosine residues, which will then serve as docking sites for the STATs. STAT proteins comprise a family of transcription factors latent in the cytoplasm that consists of seven different members in mammals; i.e. STAT1, 2, 3, 4, 5a, 5b and 6. The STATs bind to the receptors’ docking sites through their src-homology 2 (SH2) domain and are phosphorylated by the Jaks at single tyrosine residues. STATs dimerize by reciprocal phosphotyrosine-SH2 domain interactions and translocate to the nucleus. Activated STATs initiate transcription by binding as dimers to response elements in the promoters of target genes. Different ligands specifically activate different members of the Jak and STAT families. The Jak- STAT pathway is modulated by a range of regulatory proteins, which contribute to the specificity and diversity of cellular responses ^2-6.

1.1.1 Ligands and their receptors

The extracellular signaling proteins that signal through the Jak-STAT pathway bind members of various receptor families: single transmembrane receptors with an intrinsic protein tyrosine kinase domain (PTK receptors), single transmembrane receptors without kinase domain (non-PTK receptors) and seven transmembrane receptors (G-protein coupled receptors).

PTK receptors

To this group of receptors belong receptors for epidermal growth factor (EGF), platelet derived growth factor (PDGF), colony stimulating factor 1 (CSF-1), hepatocyte growth factor (HGF), basic fibroblast growth factor (bFGF), c-Kit and insulin. These receptors may activate STATs indirectly through Jak kinases or directly, as was described for STAT1 activation in vitro by PDGF or EGF receptor ^{7, 8}. STAT1 can bind directly to tyrosine residues on the EGF-R and CSF-1-R, STAT3 to EGF-R and HGF-R, and STAT5 to PDGF-R and insulin-R. Additionally STAT1, 3, 5 and/or 6 can be activated through these receptors ^{5, 9}.

Non-PTK receptors

This group of receptors lacks an intrinsic protein kinase tyrosine domain, but they signal through associated Jaks. They are also are referred to as cytokine receptors, and divided into four subtypes (class I-IV) based on similarities in their extracellular binding domains. Class I cytokine receptors include the following four families; i.e. gp130 family, IL-2 family, IL-3/ IL-5/GM-CSF family (gp140 family) and growth hormone (GH) family. All of the receptors belonging to class I contain four conserved cysteine residues, extracellular a tryptophan-serine-X-tryptophan-serine (WSXWS) motive and variable

Chapter 1

13 intracellular domains. Class II cytokine receptors only contains the interferon (IFN) family. Receptors within one family share two or more subunits that are brought together upon ligand binding ^{5, 10, 11}.

gp130 family. Receptors for interleukins-6 (IL-6) and IL-11, oncostatinM, LIF, cardiotrophin-1, G-CSF, IL-12, IL-23, leptin and ciliary neurotrophic factor (CNTF) all signal through a common β-chain called gp130. The IL-6 receptor (IL-6R) complex consists of a non-signal-transducing ligand binding IgG-like α-chain and two gp130 chains ¹². The LIF receptor is composed out of a signal transducing LIFRβ chain and a gp130. The CNTF receptor α chain associates with gp130 and the LIFβ chain. The IL-11 receptor α chain requires gp130 for high affinity binding and signal transduction. The receptors associate with either Jak1, Jak2 or Tyk2 and activate STAT3 using the motif YXXQ as STAT3 docking site.

IL-2 receptor family. IL-2 and IL-15 signal both through the IL-2 receptor, which consists of a non transducing α chain (IL-2Rα) and two signal transducing chains, IL-2Rβ or IL-2Rγ. The receptor for IL-4, IL-7 and IL-9 consists of individual α chains, and share the IL-2Rγ chain. The IL-4Rα chain can also combine with the IL-13Rα chain to form a functional high affinity receptor for both IL-4 and IL-13. The IL-2Rγ chain associates with Jak3, and IL-2Rβ, IL-7Rα and IL-9Rα chains with Jak1. IL-4Rα and IL-13Rα bind Jak1, Jak3 or Tyk2. They mainly activate STAT5 and STAT6.

gp140 family. Receptors for IL-3, -5 and GM-CSF have individual α chains and share the gp140 component, which associates with Jak2. They all signal through STAT5.

GH family. Receptors for GH, prolactin, erythropoietin (EPO) and thrombopoietin (TPO) form homodimers after ligand binding and do not share any receptor components. All members of the GH receptor family associate with Jak2 and bind to or activate STAT5 ⁹.

IFN family. The receptors contain four cysteine residues, but lack extracellular domains and bind IFN α,β,ω, limitin, IFNγ and IL-10 ^{5, 13}. Type I interferons; i.e. IFNα/β/ω and limitin signal through an interferon alpha receptor (IFNAR) complex, which consists of two subunits IFNAR-1 and IFNAR-2. Human IFNAR-1 is a 557-aa glycoprotein with a 21-aa transmembrane segment and a 100-aa cytoplasmic domain. IFNAR-2 exists in three forms; IFNAR-2a (short form), IFNAR-2b (soluble form) and IFNAR-2c (long form). IFNAR-2c is composed of 515 aa and acts as a functional receptor for IFNα/β signaling. The tyrosine kinases Tyk2 and Jak1 are associated with IFNAR-1 and IFNAR-2 respectively ^{14, 15}. IFNAR-2 can bind additionally STAT2 through tyrosine residues Y466 and Y481 on the receptor ¹⁶. Type II IFN, IFNγ binds to IFNγ receptor complex IFNGR, containing a chain 1 (α) and 2 (β). IFNGR-1 and -2 bind Jak1 and Jak2 respectively ¹⁷. Type I IFNs activate mainly STAT1, 2 and 3, type II IFN STAT1 ¹⁸.

G-protein coupled receptors

These receptors span the cell membrane seven times and they activate GTP- binding proteins. Only angiotensin has been shown to signal through the Jak- STAT pathway. Angiotensin II receptor (AT1) associates with Jak2 and activates STAT1 and STAT2 ¹⁹.

14 1.1.2 Janus kinases (Jaks)

Jaks are a family of cytoplasmic receptor associated protein tyrosine kinases required for cytokine signaling. In mammals the family consists of four members; Jak1, Jak2, Jak3 and Tyk2, which are activated by cytokine induced receptor dimerization. Once they are activated they cause the phosphorylation of tyrosine residues on the receptor cytoplasmic tails to provide docking sites for the recruitment of STATs recognizing these phosphotyrosines via their src- homology-2 (SH-2) domains, and thus mediating signaling of the activated STATs ¹³. Jak1, Jak2 and Tyk2 are expressed ubiquitously, whereas the expression of Jak3 is only expressed by myeloid and lymphoid cells ²⁰. The Jaks contain seven regions of homology, JH1-JH7. JH1 encodes for a kinase and is located at the C-terminal. The catalytic activity of JH1 is stimulated by phosphorylation of two tyrosine residues (Y1038/Y1039 in Jak1, Y1007/Y1008 in Jak2, Y980/Y981 in Jak3, and Y1054/Y1055 in Tyk2) in the domain. JH2 represents a pseudokinase domain; it has no catalytic function, however a Jak2 mutant lacking the domain was able to mediate GH signaling ²¹. A single glutamic acid to lysine substitution in the JH2 domain resulted in hyperphosphorylated and hyperactivated STAT when overexpressed in cells ²². The other Jak homology domains (JH7-JH3) are located at the N-terminal end and thought to be involved in receptor association and in determining the specificity of the Jak-receptor binding. The Jaks have clear functions in vivo as has been shown by knockout studies in mice ²³.

The Jak1 knockout mouse exhibited an early post-natal lethal phenotype, probably caused by a neurological defect due to loss of LIF function, which express itself in the inability to suckle ²⁴. Decreased responses to IL-7 resulted in impaired lymphopoiesis. Also responses to IFNs and IL-10 were diminished. Jak2 null mice die at embryonic day 12.5 caused by failure of erythropoiesis and immunological impairments ¹³. Mice lacking Jak3 suffer from defects in B-cell maturation (IL-7 mediated) and T-cell negative selection because of loss of gamma receptor chain (γC) (in IL-2R, IL-4R, IL-7R, IL-9R, IL-15R and IL21R) signaling. Also humans suffering from SCID were found to have mutations in the Jak3 gene ⁹. Tyk2 deficient mice were shown to be more susceptible to pathogens caused by defective responses to IL-12 and in less degree to IFN type I ²⁵.

1.1.3 STATs: structure and functional domains

The STAT genes have been identified in three chromosomal clusters. The genes encoding STATs 1 and 4 map to a region of mouse chromosome 1 (equivalent to human chromosome 2, q12-q33), STATs 3, 5a and 5b map to a region of mouse chromosome 11 (human chromosome 12, q13-14.1), STATs 2 and 6 map to a region of mouse chromosome 10 (human chromosome 17, q11.1-q22). STATs 1, 3, 4,5a and 5b are between 750 and 795 aa long, whereas STATs 2 and 6 are approximately 850 aa long ^{26, 27}. Despite functional differences of the individual STAT proteins, crystallography studies (an example of the crystal structure of activated STAT1 bound to DNA is shown in Figure 2) and protein sequence comparisons of STAT1, STAT3 and STAT4 display common STAT structural domains (an overview is depicted in Figure 1) ^{28, 29}.

Chapter 1

15

Figure 1. Domain structure of STAT proteins. Defined structural and functional regions of the STAT proteins: N, N-terminal domain; C-C, coiled-coil domain; DNA, DNA binding domain; LD, linker domain; SH2, SH2 domain with the arginine (R) conserved in all STATs depicted; P, phosphorylated tail segment with the tyrosine (Y) on which all STATs are activated; T, transactivation domain with the serine (S) on which STAT1, 3, 4 and 5a/b can be phosphorylated. Colors of the domains do correspond to the domain colors in the three- dimensional STAT picture (Figure 4).

The N-terminal domain is highly conserved and is required for the dimer- dimer interactions to form tetramers or oligomers of STATs. It has been shown for STAT1, 4 and 5 that they are able to form tetrameric complexes on tandemly linked GAS (gamma interferon activated sequence) motifs. The tetramerization of STATs contributes to the stabilization of the STAT-DNA binding and allows STAT proteins to bind with higher affinity, and therefore increases the transcriptional activity. The formation of this complex is thought to be mediated through protein-protein interactions involving tryptophan 37 (W37). Studies have demonstrated that a STAT1 W37F mutant was still able to form a dimer, however lost the ability to form tetrameric complexes on tandem sites on DNA, which resulted in loss of transcriptional activity ^30-32. Furthermore, STAT1 mutations of phenylalanine (F) 77 and leucine (L) 78 were shown to interfere with dimer formation ³³. The N-domain in STAT1 also enhances transcription by interaction with histone transacetylases (CBP/p300 proteins) ³⁴.

Arginine (R) 31 in the N-terminal domain of STAT1 is required for dephosphorylation of tyrosine 701 of STAT1. Methylation of R31 has shown to prevent STAT1 from binding to PIAS1. PIAS1 binding prevent STAT1 from binding DNA ³⁵. The deletion of the STAT1 N-terminal domain resulted in a mutant STAT1 protein which was constitutively phosphorylated on Tyr-701 ³⁶. STAT3 proteins lacking the N-terminal domain did show that the domain is not required for Y705 phosphorylation of STAT3 stimulated by IL-6 ³⁷.

The N-terminal domain is also involved in nuclear translocation. Activated STAT1 molecules lacking the entire N-terminal domain were not able to translocate to the nucleus ³⁸.

The coiled-coil domain is located between residues 136 and 315 and consists of four α-helices ²⁸. The coiled-coil domain of STAT1 (in particular lysine (K) 161) for example interacts with IRF9/p48, which makes part of the interferon stimulated gene factor 3 complex ³⁹. This domain of STAT3 interacts with transcription factor c-jun ⁴⁰ and STAT5a and 5b with the silencing mediator of retinoic acid and thyroid hormone receptors (SMRT) ⁴¹. Single mutations of STAT3’ aspartic acid (D) residue 170 or, to a lesser extent K177 diminishes receptor binding and tyrosine phosphorylation, and consequently dimer formation, nuclear translocation and transcriptional activation. Further mutation experiments with STAT3 showed that all four

16 coiled-coil domain helices are required for recruitment of STAT3 to the IL-6 receptor and for Y705 phosphorylation ³⁷. Furthermore, mutation studies of coiled-coil domain of STAT3 have revealed that only residues R214/R215 in the domain (but not R152/K153/R154, K161/K163, K177/K180, R197/R199 and K244/R245/R246) have influence on nuclear import ⁴².

The DNA-binding domain (approx. aa 320-480) is structurally similar to the immunoglobin-like DNA-binding domain and shows similarities to the DNA-binding domains of NFκB and p53 ²⁹. The domain determines the DNA binding specificity. The analysis of the crystal structure of phosphorylated STAT1 bound to DNA (Figure 2) suggested that there are few direct contacts between the DNA-binding domain of the DNA bases, i.e. K336, E421 and N460 are essential for the binding of the DNA bases, whereas R378, K410, E411, K413, T427 and T459 make contacts with phosphate backbone of DNA

29. The activated STAT3 dimer requires residues K340 and N466 for the binding to the bases in the response elements, aa M331, H332, V343, Q344, R382, R417, I431, V432, S465, I467 and Q469 make contacts to the DNA sugar-phosphate backbone ²⁸.

The domain is also important for nuclear translocation of the STAT dimers ⁴².

Figure 2. Crystal structure of tyrosine phosphorylated STAT1 dimer bound to DNA. Ribbon presentation of the STAT1 core dimer (aa 136-710) on DNA. The coiled-coil domain is shown in green, the DNA-binding domain in red, the linker domain in orange and the SH2 domain is shown in blue. The phosphorylated tail segment is colored purple and the DNA backbone in grey. ‘N’ and ‘C’ indicate the location of the lacking N- and C-termini in this STAT1 dimer. Reprint from a publication by Chen et al. ²⁹.

The α-helical linker domain (approx. aa 485-575) separates the DNA domain from the SH2 domain ²⁹. For STAT1 it was reported that its linker domain may play a role in transcriptional responses. Mutations (W539A, K544A and E545A, but not F506A and R512A) resulted in the lack of ability to induce transcriptional responses to IFNγ, but not to IFNα, although STAT1 phosphorylation, dimerization, nuclear translocation and DNA binding were normal in response to IFNγ ⁴³. Additional analyses of this STAT1 (K544A/E545A) mutant have suggested that the primary defect in this mutant is its abnormal DNA binding kinetics (it binds and dissociates from DNA more rapidly than wild type STAT1 protein) and inability to accumulate on DNA

Chapter 1

17 response elements, rather then a defective co-activator recruitment mechanism ⁴⁴.

The SH2 (src homology domain 2) domain is located between aa 575 and 680. It is required for the recruitment of STATs to phosphorylated receptors and for the reciprocal SH2-phosphotyrosine interactions between monomeric STATs to form dimers ⁴⁵. The binding of the STATs to the receptors occurs through the interaction of the conserved arginine in the SH2 domain (R602 for STAT1, R609 for STAT3 and R618 for STAT5) with the phosphorylated tyrosine residues located in the intracellular domains of the receptors. This interaction is highly specific and determines the STAT binding to the different cytokine receptors ^{28, 46}. This conserved arginine residue is also essential for the recognition of the single phosphate group of the phosphotyrosine to initiate dimerization of the STAT proteins required for STAT transcriptional activity ⁴⁷. Additional contacts between A641 and V642 in the SH2 domain and L706 C-terminal of the phosphotyrosine are essential in the dimer formation in STAT1. For STAT3 are these N641, M642 in the SH2 domain and F706 ²⁹.

The phosphorylated tail segment harbours the tyrosine residue (Y701 for STAT1, Y690 for STAT2, Y705 for STAT3, Y693 for STAT4, Y694 for STAT5 and Y641 for STAT6), which phosphorylation is essential for STAT dimerization through SH2 domain binding and DNA binding activity ⁴⁸. The four to five aa C-terminal of the phosphorylated tyrosine (for STAT1 Y701IKTEL, for STAT3 Y705LKTKF and for STAT5a Y694VKPQI) make the contacts with other aa of the SH2 domain and are therefore essential in dimerization ^{28, 29, 49}.

The transcriptional activation domain (TAD) at the C-terminal region between approximately aa 700 and 851, is involved in the communication with transcriptional complexes. A conserved serine in STAT1 (S727), 3 (S727), 4 (S722) and 5 (S726/731) can be phosphorylated and regulates STAT transcription. Several studies showed an reduced transcriptional activity when this serine is replaced by alanine ^50-52. Alternative splicing at the 3’end of the gene transcripts generates shorter isoforms of STAT1, 3, 4, 5a and 5b. The isoforms lack the functional transcriptional activation domain, but are still able to bind to the response elements of target genes. When the isoforms are overexpressed, they can act as dominant negative regulators of transcription, by competing with full length STATs for the DNA binding sites ⁵³.

1.2 Mechanisms and regulation of Jak-STAT signaling 1.2.1 IFN signaling

Type I interferons are produced by leukocytes and fibroblasts upon virus infection and type II IFN is produced by activated Thelper (Th)1-cells and natural killer (NK) cells ^{54, 55}. IFNα/β (type I) gene transcription in virus infected cells is an important early event in the host response to infections.

IFNα/β is produced by virus-infected cells within hours and plays an important role in preventing virus spread. Production of IFN-gamma on the other hand needs activation of the immune system ⁵⁶. The induction of transcription of IFNα/β genes is mediated through the binding of interferon regulatory factors (IRF) to virus-inducible enhancers in their promoter

18 regions ¹⁷. IRF3 and IRF7 are the key regulators of the induction of IFNα/β genes. IRF3 is phosphorylated and translocated to the nucleus upon viral infection. It associates with the co-activator p300/CBP, and activates IFNβ and IFNα1 genes. IFNα and β are produced, bind to their receptors, signal through the Jak-STAT pathway and activate a number of target genes, collectively called interferon stimulated genes (ISGs). One of them is IRF7. In the presence of viral infection IRF7 is also phosphorylated, translocates to the nucleus, forms a dimer with IRF3 and transcriptionally stimulates additional IFNα genes ^{57, 58}.

Signals of both types IFN are transmitted through the Jak-STAT pathway, as illustrated in Figure 3. Binding of the IFNs to their receptors links the two components of the receptors and results in the activation of Jak1 and Tyk2 for IFNα/β, and Jak1 and Jak2 for IFNγ. The two kinases (Jak1/Tyk2 and Jak1/Jak2) transactivate each other by transphosphorylation. The activated kinases then phosphorylate tyrosine residues on the receptors: Y440 on IFNGR-1 and Y466 on IFNAR-2, which are then able to bind STAT1 and STAT2 respectively through their SH2 domains on these sites. As a result STAT1 is phosphorylated by the Jaks on its tyrosine 701, STAT2 on Y690 and STAT3, which is also activated by IFNα/β, on Y705.

Figure 3. Schematic overview of IFNα/β and γ induced signal transduction through the Jak-STAT pathway. The Jak1 and Tyk2 kinases are activated by IFNα/β, which leads to phosphorylation, dimerization and nuclear translocation of STATs. IFN type I activates two classes of STAT complexes. STAT1 (p91) and STAT2 (p113) heterodimer combine with IRF9 (p48) to form ISGF3, which activate transcription of ISGs through ISRE and STAT1 and STAT3 homodimers, which bind to GAS elements. Upon IFNγ binding are STAT1 homodimers formed, which induces transcription by binding to GAS element ⁶⁰.

Chapter 1

19 Through reciprocal phosphotyrosine-SH2 domain interactions STAT1 forms homodimers (IFNα/β/γ), STAT3 forms homodimers and heterodimers with STAT1 (IFNα/β) and additionally upon IFNα/β signaling STAT1 and STAT2 are able to form a heterotrimeric complex with IRF-9 (p48) , designed interferon stimulated gene factor 3 (ISGF3). The STAT dimers will translocate to the nucleus and to the specific response elements (STAT1 and 3 homo and heterodimers to GAS elements, and ISGF3 to ISRE) in promoters of target genes and stimulate transcription ⁵⁹.

IFNs have antiproliferative, immunomodulatory and antiviral activity. Mice lacking IFNAR1 have increased susceptibility to vesicular stomatitis virus (VSV), Semliki Forest virus SFV), vaccinia virus or lymphocytic choriomeningitis (LCMV). In contrast mice deficient for IFNGR1 were able to overcome a VSV and SFV infection, died from vaccinia virus infection and were increased susceptible, however less than the IFNAR KO mice, to LCMV.

Furthermore IFNAR KO mice were resistant against the pathogen Listeria monocytogenes, whereas the IFNGR deficient mice were highly susceptible.

This points out that IFN type I and II have partially overlapping but distinct antiviral effects ^{61, 62}. Differences in the induction of target genes by IFN type I and II are responsible for the differences in antiviral effects. Especially the use of oligonucleotide arrays to study the differential expression of mRNA in response to IFNs have identified many new and confirmed known interferon- stimulated genes and provided insights into the mechanisms of IFN action ^63,

64. IFNα/β induced genes (i.e. ISG) include ISG-56K, MxA, 6-16, IRF1, 2’-5’- oligoadenylate synthetase (OAS), double stranded RNA activated protein kinase (PKR), eIF-2α, MHC-class I, IL-12, IL-15, ICE and IFNα/β. Antiviral activity requires induction of OAS, PKR and Mx proteins ⁶⁵,¹⁵. Direct antiviral effects of IFNγ are mediated through its upregulation of PKR, OAS and dsRNA specific adenosine deaminase (dsRAD) ⁵⁵. IFNγ has direct antibacterial activity through the activation of monocytes, lymphocytes, neutrophils and macrophages by the enhancement of expression of chemokines, inducible nitric oxide synthase (iNOS) and natural resistance-associated macrophage protein1 (NRAMP1) ^{55, 65}.

IFNs are also modulators of the immune system and do influence cell growth and differentiation. IFNα/β suppress the antigen-specific- or mitogen-induced proliferation of T helper cells and cytotoxic T cells on one hand, while at the other hand they induce IL-15 production in macrophages, which enhances T- cell growth and proliferation of NK-cells. IFNα/β also induce expression of MHC classI, thereby stimulating antigen presentation ^{15, 61}.

IFNγ is a strong inducer of both MHC class I and II molecules. ⁵⁵. IFNγ can also regulate the isotypes of immunoglobulins secreted during humoral immune responses ⁶³ and induces cell death of T-cells ⁵⁴.

IFN signaling is also involved in apoptosis and in the regulation of cell cycle.

IFN induced growth inhibiting and proapoptotic genes included IRF1, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), XIAP-associated factor 1 (XAF1), galectin 9, a cyclin E binding protein, amphiphysin 1, MyD88, and several ubiquitin pathway genes ^{15, 55, 64}.

20 1.2.2 IL-6 signaling

IL-6 is a cytokine that exhibits a wide range of biological effects. It is induced by proinflammatory stimuli like IL-1 and TNFα, which are expressed at most inflammatory sites. ¹¹ TNFα activation leads to activation of NFκB, which is the transcription factor for the induction of IL-6 gene expression. In liver, TNFα activates IL-6 expression mainly in Kupffer cells, but also in endothelial cells and hepatic stellate cells, resulting in Jak-STAT/ SHP2/MAPK activation in hepatocytes ^66-68.

Signaling through the Jak-STAT pathway is illustrated in Figure 4. IL-6 binds specifically to the IL-6R α-chain, which allows recruitment of the two signaling gp130 receptor subunits. The receptor subunit gp130 is ubiquitously expressed, but the number of cells responding to IL-6 is limited because of the restricted availability of the α-chain. The soluble form of IL-6Rα (sIL-6Rα) can as well be used to initiate IL-6 signaling when bound to IL-6. In addition to sIL-6Rα, also soluble gp130 (sgp130) is present in serum. Soluble gp130 is probably translated from an alternative spliced mRNA and can neutralize IL- 6/sIL-6Rα complexes, thereby acting as an antagonist. IL-6 alone is not able to interact or cause homodimerization of gp130 ^{12, 69-71}.

Figure 4. Schematic diagram of IL-6 induced signaling. IL-6 activates the Jak-STAT pathway and the MAPK cascade. IL-6 binding to the IL-6 receptor causes aggregation of the three receptor chains and consequently activation of Jak1, Jak2 or Tyk2. The receptor chain gp130 is phosphorylated on four tyrosine residues (Y767, Y814, Y905 and Y915) to allow binding of STAT3. Activated STAT3 forms homodimers, which are binding to GAS-like response elements. Through activation of SHP2 is IL-6 also coupled to MAPK signaling (TF, transcription factor) ⁷⁰.

As result of the receptor-IL-6 binding, gp130 associated kinases Jak1, Jak2 and Tyk2 become phosphorylated and will in return phosphorylate six tyrosine residues on the cytoplasmic tail of the gp130 receptor chains. Four of these tyrosine residues serve as docking sites for the SH2 domains of STAT3 (Y767, Y814, Y905 and Y915) and of STAT1 (Y905 and Y915) ⁷⁰, which will subsequent be phosphorylated on their single tyrosine residues (Y705,

Chapter 1

21 respectively Y701). The phosphorylated STAT1 and 3 will form homo and heterodimers and translocate to the nucleus to bind to GAS-like response elements.

Activation of the IL-6R complex does not only lead to signaling through the Jak-STAT pathway, but also to activation of the SHP2/MAPK signaling pathway. One of the six tyrosine residues (Y759) on gp130 cytoplasmic tail, is bound by SHP2 (SH2 domain containing tyrosine phosphatase). Upon Jak- SHP2 interaction, SHP2 will be phosphorylated by Jak1 on its tyrosine residues 304 and 327. Subsequently Grb2 (growth-factor-receptor-bound protein), which is linked to SOS (son of Sevenless), can bind to the pY304 of SHP2. Recruitment of SOS to the receptor complex at the membrane allows Ras activation. This leads to signaling through the Ras-Raf-MAPK cascade ^12,

70. Activated MAPK activates the transcription factors NF (nuclear factor)-IL6, and CAAT/enhancer binding protein (C/EBP) β and δ, which bind to the promoters of acute phase proteins to induce transcription ^{11, 72}. Through association of SHP2 with Gab1 (a scaffolding adaptor protein) ERK2 is activated. This interaction links IL-6 signaling to the activation of the PI3K /Akt pathway which mediates anti-apoptotic effects and cell growth ^{12, 70}.

SHP2 not only links IL-6R complex activation to the MAPK pathway, it also has an inhibitory role. Through its activation and association with Y759 on the receptor, it counteracts receptor and STAT activation by tyrosine dephosphorylation of receptor kinase sites, resulting in inhibition of IL-6 induced gene expression. Furthermore, IL-6 signaling is negatively regulated by SHP1, which can dephosphorylate Jak2 and Tyk2, and by SOCS3, which competes with SHP2 for binding of Y759 on the IL-6R complex and inhibits Jak activity ^{71, 73, 74}.

Il-6 acts on various cells. In addition to its important role in inflammation, Il- 6 induces differentiation and development of haematopoietic cells, osteoclasts, neural cells and hepatocytes. It also acts as growth factor for renal cell carcinoma and Karposi’s sarcoma, and promotes growth of haematopoietic stem cells ⁶⁷.

IL-6 deficient mice develop normally, probably because other members of the gp-130 cytokine family do compensate for the lack of IL-6. However they fail to control efficiently infection with vaccinia virus or Listeria monocytogenes and have an impaired T-cell dependent antibody response against vesicular stomatitis virus. Most important, the inflammatory acute-phase response after minor tissue damage or infection with bacterial lipopolysaccharide (LPS), was severely impaired ^{75, 76}. Furthermore do they show a decreased IL-10 production and increased IL-12 production in macrophages.

IL-6 target genes involved in anti-inflammatory responses include IL-1R antagonist, soluble TNFα-R, IL-10, acute phase proteins, glucocorticoids, tissue inhibitor of metalloproteinase-1 (TIMP-1) and SOCS3 ^{66, 77}.

1.2.3 Nucleocytoplasmic transport

In unstimulated cells STATs predominantly localize to the cytoplasm, and upon stimulation rapidly translocate to the nucleus and induce gene expression. Nuclear translocation is mediated by the nuclear pore complex (NPC).

Active nuclear import is directed by a nuclear localization signal (NLS), which is recognized and bound by members of the importin family. Importin-α5

22 recognizes the NLS and functions as an adaptor for the binding of importin-β.

Importin-β interacts with the NPC and mediates the transport of the STAT dimer into the nucleus. Hydrolysis of GTP by Ran, a Ras-like small GTPase, provides the energy that is required to translocate the STATs through the central gated channel of the NPC. NLS elements include a short stretch arginine and lysine residues. Mutagenesis studies of possible NLS motifs have indicated that K410-R418 in STAT1 and R409-K415 in STAT2 are required for IFN-induced nuclear import ⁷⁸. Also a single mutation in STAT1 leucine 407 (L407A), or double mutations in lysines 410 and 413 (K410A/K413A), were shown to block nuclear import due to abolishment of the importin-α5 binding to these mutated STAT1 molecules. The L407 mutant can still bind DNA, whereas the K410A/K413A mutant can not ⁷⁹. Mutations of R414/417 in STAT3, corresponding to K410/413 in STAT1, also caused loss of nuclear import ability of STAT3 ⁴².

Nuclear export is specified by nuclear export signal (NES) elements. Three nuclear export signal (NES) elements have described for STAT3; i.e. aa306- 318, aa404-414 and aa524-535, and for STAT1; aa302-314, aa399-410 and aa534-533^{80, 81}. The NES element is recognized by an exportin CRM1 (chromosome region maintenance 1), which is bound as well to Ran-GTP in the nucleus. The CRM1-STAT-Ran/GTP complex is exported through the NPC and dissociates in the cytoplasm after the hydrolysis of Ran-GTP ^{13, 81}.

1.2.4 STAT DNA binding and transcriptional activation

Activated STATs can bind to two classes of DNA response elements in promoters of target genes. The STAT dimers, STAT1, 3, 4, 5 and 6 homodimers and STAT1 and 3 heterodimer bind to variations of GAS (gamma activated sequence)-like motifs, palindromic response elements TTCNNNGAA, except for STAT6 which prefers TTCNNNNGAA ⁸². GAS-like elements have been identified in promoters of many different genes, like for cFos, M67, FcγR1, IRF1 and β-casein. The other class of response elements where STATs bind to are interferon stimulated response elements (ISRE); i.e.

AGTTTNNNTTTCC. ISRE elements are bound by ISGF3 (interferon stimulated gene factor 3); i.e. the complex of STAT1 and STAT2 together with IRF9/p48 (interferon regulatory factor 9) ⁸³. These elements can be found in ISG like ISG54, ISG15, 6-16 and OAS ⁴.

Once the activated STAT dimer binds to its target promoter, the transcription rate from this promoter is increased. The STATs have the ability through their TAD to recruit nuclear co-activators that mediate chromatin modifications and communication with the promoter. The TAD of STAT1 can directly interact with the CREB-binding protein (CBP)/p300 family of transcriptional coactivators to contribute to transcriptional activation.

Recently was shown that both tyrosine-phosphorylated STAT1α (full-length wild-type protein) and STAT1β (lacking the TAD) stimulate in vitro transcription on a naked DNA template. Using a system with purified proteins and naked DNA, it was shown that STAT1α- and STAT1β-dependent transcription is stimulated by the TRAP/Mediator co-activator complex.

Although both STAT1α and STAT1β bind to known STAT sites within in vitro assembled chromatin templates, only STAT1α, and not STAT1β, in cooperation with p300 and acetyl-CoA, stimulated in vitro transcription from chromatin. After IFNγ treatment, it was shown that cells recruit STAT1α or -β

Chapter 1

23 to the chromosomal interferon-1 gene, but only STAT1α-containing cells recruit p300 and stimulate transcription. The results indicate that the recruitment of p300 to the chromatin is required to make TRAP/Mediator effective in stimulating transcription ^{34, 84}.

Furthermore, transcriptional activity can be positively regulated by phosphorylation of the conserved serine residue in the TAD. Serine phosphorylation is independent of tyrosine phosphorylation ⁸⁵ and is not required for DNA binding of the activated STATs⁸⁶. It has been described that an activated JAK2 is the kinase responsible for serine phosphorylation ⁸⁵; other candidates include p38, ERK and JNK ⁸³. Expression of a dominant- negative p38 led to defective STAT1 serine 727 phosphorylation and diminished IFNγ mediated protection against viral killing ⁸⁷. In contrast have Zhu et al. showed that ERK, JNK and p38 do not exhibit kinase activity under IFNγ stimulation ⁸⁵.

1.2.5 Negative regulation

Inactivation or inhibition of Jak-STAT activation is important in the regulation of their biological signaling effects. As result of actions of negative regulators at several points in the signaling pathway, the duration of STAT activation is temporary. After STATs are phosphorylated, dimerized, translocated to the nucleus and have induced transcription, they are exported back to the cytoplasm in dephosphorylated state.

Factors involved in negative regulation are members of the SOCS (suppressors of cytokine signaling) family, of the PIAS (protein inhibitor of activated STATs) family, PTPs (protein tyrosine phosphatases) and enzymes that mediate modifications of STATs proteins, like ubiquitination and SUMO- ylation^{13, 23}. At the receptor level the pathway is negatively regulated through receptor internalization to endocytic vesicles ⁸⁸.

SOCS

They comprise a family of eight members who share a similar structure, with a central SH2 domain, a region of homology called the SOCS box at the C- terminal and a kinase inhibitory region located at the N-terminal. Members are CIS-1 (cytokine-inducible SH2 containing protein) and SOCS1-7 ⁸⁹. SOCS are target genes of the STATs and do thus generate a negative feedback loop.

They inhibit STAT signaling by binding to the phosphorylated tyrosine residues on the receptors to physically block binding of STATs, by binding of Jaks or to the receptors to inhibit Jak kinase activity or by interacting with the elongin BC complex and cullin 2 to induce ubiquitination of the Jaks ^{6, 89}. Ubiquitination of the Jaks prepares them for proteasomal degradation ⁵. The essential negative regulatory function of SOCS1 was revealed by the fatal, immune-mediated inflammatory disease which is present in SOCS 1 deficient mice ²³.

PIAS

This family contains five members, PIAS1, PIAS3, PIASγ, PIASxα and PIASxβ.

PIAS1, PIAS3 and PIASx can specifically interact with STAT1, STAT3 and STAT4, respectively. PIAS-STAT interaction is cytokine dependent. PIAS family members have a central Zn-binding RING-finger domain, a well conserved domain at the N-terminus and a less-well conserved C-terminal

24 domain. PIAS bind to phosphorylated STAT dimers and prevent them from binding DNA ⁹⁰. Inhibition of R31 methylation of STAT1 proteins, by methylthioadenosine (MTA), a methyl-transferase inhibitor promotes PIAS1 binding and thus inhibits STAT1 signaling. With other words, arginine methylation protects the STATs from binding to PIAS1 and deactivation. The methylation of the highly conserved R31 residue in STAT1 by the protein arginine methyl-transferase PRMT1 was found to be required for IFN type I induced transcription ³⁵. PIAS 1 binding of STAT1 seems to protect STAT1 from dephosphorylation by TC-PTP/TC45. So arginine methylation of STAT1 regulates STAT1’ dephosphorylation ⁹¹. In addition to PIAS1, PIASy also interacts with STAT1 and seems to function as corepressor of STAT1.

Very recently the PIAS1 KO mouse has been reported by Liu et al. Tyrosine phosphorylation of STAT1 induced by IFNα or IFNγ and likewise of STAT2 by IFNβ, was not altered in PIAS1-/- cells. Gene activation analysis showed that PIAS1 selectively regulates a subset of IFNγ or IFNβ target genes. Due to PIAS1 inhibitorial function, the immune response to viral and bacterial infections was enhanced in these PIAS1 deficient mice ⁹².

It has been reported that PIAS proteins have E3 ligase activity for SUMO- ylation mediated by the RING finger domain. However in the PIAS1 -/- thymocytes no defect was obeserved in SUMO3 or SUMO1 protein modification ⁹². SUMO (small ubiquitin-related modifier) has been found added to target proteins by special protein-protein interactions. This is regulated by SUMO E3 ligases ⁹³. How SUMO-ylation of STAT inhibits its activation is not yet understood.

Protein tyrosine phosphatases

PTPs are active both in the cytoplasm and the nucleus. SHP1 and SHP2 are cytoplasmic PTPs, which can dephosphorylate Jaks and receptors. SHP1 has been shown to directly bind and dephosphorylate Jak2 ⁸⁹. Also SHP2 seems to be a regulator of Jak2 activity. Dephosphorylation of Jak2 by SHP2 protects Jak2 from SOCS1 mediated ubiquitination and degradation ⁹⁴. PTP1b specifically inhibits JAK2 and Tyk2 ²³. Although TC-PTP/TC45 (T-cell protein tyrosine phosphatase 45 kDa isoform) is known as a nuclear PTP, there is also evidence that it is involved in the dephosphorylation of Jak1 and Jak3. It was shown that it can bind to Jak1 and Jak3. Furthermore cells from mice deficient for TC-PTP/TC45 show increased phosphorylation of Jak1, STAT1 and STAT5 depending on the stimulation. Jak2 phosphorylation was not affected ⁹⁵. Nuclear tyrosine phosphatases TC-PTP/TC45 was identified to dephosphorylate specifically phosphorylated STAT1 in the nucleus. In TC-PTP null mouse embryonic fibroblasts (MEFs) the dephosphorylation of STAT1 is defective, as well as the dephosphorylation of STAT3, but not of STAT5 and 6.

Reconstitution with TC-PTP/TC45, but not with TC48, the other TC-PTP isoform, could repair this defect ⁹⁶.

1.2.6 Specificity and diversity in signaling

Diversity and specificity are decisive factors in the determination of the outcome response in the complexity of intercellular signaling. The different cytokines induce specific and distinct biological responses in different types of cells by activating different members of the Jak and STAT families. However, even in the same type of cell, the responses to a certain cytokine can vary depending on the location of the cell and the condition of its

Chapter 1

25 microenvironment. The cell specificity of cytokine action through the Jak- STAT pathway is determined at several levels, which are obviously influenced by one another ⁹⁷.

To start with, specificity is reached through the selective activation of the different STATs by specific ligands. This is achieved through the selective binding of different STAT family members to phosphotyrosine docking sites on receptors ^{46, 98}. This was shown by domain swapping experiments the docking sites ⁹⁸ and with the STAT SH2 domains ⁴⁶. STAT2 constructed with the SH2 domain of STAT1 could be activated by IFNγ, whereas the STAT1 constructed molecule including the STAT2’ SH2 domain could not. When the tyrosine residues which phosphorylation is required for dimerization and nuclear translocation of the STATs, with surrounded sequence of STAT1 (Y701) and STAT2 (Y690) were swapped, the STAT1’SH2 domain was required to induce phosphorylation of the STAT protein by IFNγ ⁴⁶. Using a phosphopeptide library to determine the sequence specificity of the receptor binding sites (i.e. peptides) for the SH2 domains of STAT1 and STAT3, did show by Wiederkehr-Adam et al. that STAT1 preferentially binds peptides with a motif phosphotyrosine-(aspartic acid/glutamic acid)- (proline/arginine)-(arginine/proline/glutamine), whereby a negatively charged aa at +1 excludes a proline at+2 and vice versa. STAT3 preferentially binds peptides with a motif phosphotyrosine-(basic or hydrophobic)-(proline or basic)-glutamine ⁹⁹.

Another level of specificity is determined by the specific binding of activated STAT dimers to slightly different response elements. Variations of the inner nucleotides between the different palindromic GAS-like motifs, have been shown to influence the binding affinity towards specific STAT dimers.

For example STAT1 homodimer binds to the GAS element in the promoter of the FcγR1 gene, whereas the STAT3 homodimer does not ¹⁰⁰. Furthermore, it seems that the ability of STATs to bind cooperatively to tandem GAS elements (which are 6-10 bp apart) also contributes to DNA binding specificity ^{82, 101}.

1.3 Biological functions of STAT proteins

The biological role of each individual STAT protein has been examined through the study of knockout mice.

STAT1

STAT1 KO mice are highly susceptible to microbial and viral infections and tumor formation due to defective IFN responses. These mice failed to induce transcription of STAT1 target genes in response to both IFNα/β and IFNγ.

They also failed to induce ISGs in response to IFNα ¹⁰². It is also demonstrated that STAT1-deficient mice exhibit a higher incidence of spontaneous and chemically induced tumors. This can be explained through STAT1 roles in upregulation of proapoptotic genes (caspases¹⁰³) and its role in the regulation of expression of proteins involved in antigen presentation (MHC class II) and thus affecting the immunogenicity of the tumor ¹⁰⁴. Furthermore, it is shown that STAT1 interacts with p53 to enhance DNA damage-induced apoptosis. In addition, STAT1 could suppress Mdm2 protein in STAT1-deficient cells.

Mdm2 protein interacts with p53 to induce its degradation ¹⁰⁵.

26 STAT2

STAT2 is the only member that does not bind to GAS elements. Its biological function is not well understood sofar, however since it is activated by IFNα/β it is clear that it plays a critical role in promoting antiviral immune response.

STAT2 knockout mice are, like STAT1 KO susceptible to viral infections.

Studies in these mice suggested that STAT2 regulates the basal levels of STAT1 expression in MEFs, since upregulation of STAT1 in response to IFNα is impaired. This was not the case in macrophages ¹⁰⁶. STAT1/STAT2 double knockouts are completely unresponsive to all IFNs and show more susceptibility to infection than either of the single knockouts.

STAT3

STAT3 is activated by many cytokines. STAT3 KOs are embryonically lethal at day 7.5. However the generation of tissue-specific knockouts using the Cre-lox method has revealed STAT3’ role in a wide variety of tissues ¹⁰⁷. The lack of STAT3 in skin showed that it is important in the migration of keratinocytes and is essential during the hair cycle and wound healing ¹⁰⁸. STAT3 deficient T-cells exhibit loss of proliferative response to IL-6, which has previously been shown to suppress apoptosis in these cells ¹⁰⁹. Furthermore do STAT3- deficient T-cells show an impaired IL-2-mediated IL-2 receptor (IL-2R) alpha chain expression ¹¹⁰. The lack of STAT3 in liver results in an impaired acute phase response, caused by a defective STAT3 induced expression of fibrinogen, haptoglobin, serum amyloid (SAP), serum amyloid A1 (SAA) and proteinase inhibitor α2-macroglobulin ⁷². STAT3 does also play an essential role in the normal glucose homeostasis by downregulation of the expression of gluconeogenic genes like phosphoenolpyruvate carboxykinase-1 (PCK-1) and glucose 6-phosphatase (G6PC). Mice with liver-specific STAT3 deficiency showed insulin resistancy associated with increased hepatic expression of PCK-1 and G6PC genes ¹¹¹. Liver partial hepatectomy in STAT3 liver-specific KO mice showed that STAT3 promotes cell cycle progression and cell proliferation during liver regeneration. Activation of MAPK signaling was normal in these livers ¹¹². In thymus, it impairs the postnatal maintenance of thymic architecture and thymocyte survival ¹¹³. Mice deficient for STAT3 in macrophages and neutrophils are more susceptible to endotoxin shock with an increased production of inflammatory cytokines such as TNF alpha, IL-1, IFN gamma, and IL-6 by the macrophages and neutrophils probably due to impaired IL-10 suppressive effects on this production in these mice ¹¹⁴. Deletion of STAT3 in mammary glands results in delayed apoptosis that occurs during cyclical mammary gland involution ¹¹⁵. Selective targeting of the full length STAT3α and truncated STAT3β splice variants showed specific functions for both isoforms. Mice lacking STAT3β are hypersensitive to endotoxin-induced inflammation ¹¹⁶. In liver-specific STAT3 isoform KO mice it was reported that STAT3β is not a dominant negative factor, since its expression can rescue the embryonic lethality of a complete STAT3 deletion.

Both isoforms can induce acute phase proteins. STAT3α however is responsible for regulation of the IL-6 response and of the IL-10 anti- inflammatory function in macrophages ¹¹⁷.

Chapter 1

27 STAT4

Since Il-12 activates STAT4, the STAT4 knockout mouse does display defective IL-12 responses; i.e. impaired T-helper-cell type I (Th1) differentiation and therefore impaired IFNγ production and cell-mediated immunity ¹¹⁸. Because of the defective Th1 response these animals are resistant to Th1 mediated autoimmune diseases like arthritis and diabetes ¹¹⁹.

STAT5

The two STAT5a and STAT5b proteins are approximately 95% identical in aa sequence. Mice deficient for only STAT5a ¹²⁰ or STAT5b ¹²¹ have revealed specific and limited functions for the two STAT5 proteins individually ¹²². Through analyses of these single knockouts has been revealed that STAT5a is important in prolactin, GM-CSF and IL-2 signaling. STAT5a KO mice show no effect on body growth or serum IGF-1 (mediator of the somatic effects of GH) levels ¹²². In liver is STAT5a, together with STAT5b the most important mediator activated by GH ¹²³. STAT5b is important in controlling somatic growth in mice. It is as well regulating sexually dimorphic gene patterns, which exhibits itself in STAT5b KOs by GH mediated loss of male- characteristic body growth rates and male specific liver gene expression of cytochrome P450 (CYP) steroid hydroxylases. Levels of male specific CYP2D9 and testosterone 16α-hydroxylase were decreased in these knockouts and levels of female liver specific CYP3a, CYP2B and testosterone 6β-hydroxylase were increased. In female mice are both STAT5a and STAT5b required for the constitutive expression of hepatic female specific GH-regulated, CYP2B, as well as several CYP-catalyzed testosterone hydroxylase activities ^{124, 125}.

STAT5a is furthermore required for mediating prolactin signaling and mammary gland development and lactogenesis ¹²⁰. STAT5a/5b double knockout females are infertile, caused by an altered corpus luteum development. This is in contrast to single STAT5a or 5b deficient mice, which are fertile, suggesting that the function of the STAT5 proteins may be redundant ¹²².

STAT6

STAT6 is required for the induction of IL-4 dependent gene expression leading to type II T-helper-cell (Th2) differentiation and IL-4 dependent B- cell proliferation. In addition, has it been demonstrated that STAT6 is important for the functions mediated by IL13, which is related to IL4. IL4 and IL13 have been shown to induce the production of IgE, which is a major mediator in an allergic response. STAT6 activation might therefore be involved in IL4- and IL13-mediated disorders such as allergy ¹²⁶¹²⁷. This was proven in STAT6 deficient mice, which display a reduced pathology in asthma.

Furthermore is it described that STAT6 deficiency is associated with enhanced tumor immunity, since natural killer T-cells (NKT-cells) and IL-13, produced by NKT-cells and signaling through the IL-4R-STAT6 pathway, are necessary for down-regulation of tumor immunosurveillance ¹²⁸.

28 1.3.1 Impact of STATs in disease

Since STAT proteins display a wide range of functions in many biological aspects, they are consequently involved in or mediating pathological processes.

Many viruses have developed mechanisms to inhibit the anti-viral immunity actions of IFNs through disruption of IFN induced Jak-STAT signaling.

Hepatitis C virus (HCV) inhibits IFN signaling downstream of STAT phosphorylation ¹²⁹. Duong et al. showed that acceleration of the negative feedback loop of Jak-STAT signaling, through an increased upregulation of PP2Ac, is responsible for decreased binding of activated STAT1 to the DNA and inhibition of signaling ¹³⁰. In vitro and in vivo studies to demonstrate HCV interference with IFNα induced Jak-STAT signaling are described in chapter 2 and 3. Interference with IFN induced Jak-STAT signaling in many different ways as mechanism of viral persistence, is also reported for other viruses as simian virus 5 ¹³¹, herpes simplex virus type 1 (HSV-1) ¹³², hepatitis B virus (HBV) ¹³³, Sendai virus ¹³⁴, human parainfluenza virus type 2 ¹³⁵, adenovirus

136, 137, human papilloma virus (HPV) ^{138, 139}, Epstein-Barr virus (EBV) ¹⁴⁰, cytomegalovirus (CMV) ¹⁴¹ and paramyxoviruses like mumps virus ^{142, 143}, measles virus ¹⁴⁴ and Nipah virus ¹⁴⁵.

Other examples of diseases caused by a defective signal transduction through the Jak-STAT pathway are X-linked combined immunodeficiency (XCID) and X-linked severe combined immunodeficiency (XSCID). In both diseases Jak3 activation is impaired due to mutations of the IL-2Rγ chain. C- terminal truncations of this receptor chain completely prevent Jak3 binding and results in development of XSCID. In XCID the Jak3 association with the receptor is only reduced due to a point mutation in IL-2Rγ chain. Mutational Jak3 deficiency has also been detected in patients with the less severe form SCID ⁹¹⁴⁶.

STAT3 might be involved in the pathogenesis of Crohn’s disease. Mice with tissue-specific deletion of STAT3 during haematopoiesis showed Crohn’s disease-like pathogenesis and intestinal T-cells from patients with Crohn’s disease express constitutively activated STAT3 ^{147, 148}.

Dysregulation of STAT signaling pathways has been demonstrated to contribute malignant cellular transformations by promoting cell cycle progression and/or cell survival. Constitutive activation of Jak and STAT proteins has been reported in a large and growing number of malignant cell lines and human cancers ^{5, 149}. Constitutive activation of STATs is often associated with malignant transformation induced by oncogenes coding for oncogenic tyrosine kineses like members of the Src, Abl or Ras family. Also a constitutive active STAT3 (STAT3-C; substitution of two cysteine residues in the SH2 domain) could function as oncogene independently of a tyrosine kinase oncogene. This molecule is capable of driving transcription and induces cell transformation ¹⁵⁰. Constitutative activated STAT3 and STAT5 participate in oncogenesis through upregulation of genes encoding apoptosis inhibitors like Mcl-1, Bcl-XL and Bcl-2 to promote cell survival ^{151, 152} and cell cycle regulators like cyclins D1, 2, 3 and A, c-myc, cdc25A to promote cell cycle G1 to S phase transition ^{153, 154} and inducers of angiogenesis like VEGF ¹⁵⁵.